Chromatography is widely used in separation and analysis of mixtures of compounds. Due to limitations in peak capacity of one-dimensional chromatography, multi-dimensional chromatography systems with significantly increased peak capacity have been devised for the analysis of complex samples. Two-dimensional (2D) chromatographic techniques have become very popular especially in the analysis of complex mixtures. As compared to one-dimensional (1D) chromatography, 2D chromatographic techniques have higher selectivity and resolving power assuming the retention mechanisms are complementary. Maximum peak capacity in a two-dimensional separation system is achieved when the selectivity of the individual separations are independent (orthogonal), so that components which are poorly resolved in the first dimension may be completely resolved in the second dimension. If orthogonal separation mechanisms are used in the two dimensions, the theoretical peak capacity of the system is the product of the individual peak capacities. See Giddings, J. C. Anal. Chem. 1984, 56:1258A; Giddings, J. C., in: Cortes, H. J. (Ed.), Multidimensional Chromatography: Techniques and Applications, Marcel Dekker, New York 1990, p. 1; Jandera, P. et al., Chromatographia 2004, 60:S27.
However, some restrictions exist for 2D chromatography in terms of sensitivity and solvent compatibilities. For example, the mobile phase that is carried over from the first dimension often creates interference with the second dimension, thus limiting the separation capability of the second dimension. The incompatibility of solvents used in the first and second dimensions can cause severe band dispersion or broadening and peak deterioration, thus posing a big challenge for the interface design. Tian, H., et al., J. Chromatogr. A 2006, 1137:42. To alleviate the solvent immiscibility concern, researchers have developed several 2D systems that use compatible mobile phases in both dimensions. Some examples include the following: 2D Reversed Phase Liquid Chromatography (RPLC×RPLC) (Venkatramani, C. J. et al., J. Sep. Sci. 2012, 35:1748; Zhang, J. et al., J. Sep. Sci. 2009, 32:2084; Song, C. X. et al., Anal. Chem. 2010, 82:53; Liu, Y. M. et al., J. Chromatogr. A 2008, 1206:153), 2D Hydrophilic Interaction Liquid Chromatography (HILIC×RPLC) (Liu, Y. M. et al., J. Chromatogr. A 2008, 1208:133; Wang, Y. et al., Anal. Chem. 2008, 80:4680; Louw, S. et al., J. Chromatogr. A 2008, 1208:90), 2D Normal Phase Liquid Chromatography×Supercritical Fluid Chromatography (NPLC×SFC) (Gao, L. et al., J. Sep. Sci. 2010, 33:3817), and 2D SFC×SFC (Zeng, L. et al., J. Chromatogr. A 2011, 1218:3080; Lavison, G. et al., J. Chromatogr. A 2007, 1161:300; Hirata, Y. et al., J. Sep. Sci. 2003, 26:531; Okamoto, D. et al., Anal. Sci. 2006, 22:1437; Guibal, P. et al., J. Chromatogr. A 2012, 1255:252). Anderer et al., US 2013/0134095 disclosed a 2D LC system and methods which attempt to further reduce the interference with the second LC by the first LC by controlling the injection event of injecting an output of the first LC into the second LC in relation to the state of the second LC.
One technique that couples the incompatible “normal phase” and “reversed-phase” dimensions is 2D SFC×RPLC (Francois, I. et al., J. Sep. Sci. 2008, 31:3473-3478; Francois, I. and Sandra, P. J. Chromatogr. A 2009, 1216:4005). In this case, the non-polar supercritical carbon dioxide in the SFC fractions is evaporated off (when exposed to atmospheric pressure) to yield fractions with compatible mobile phases to the second RPLC dimension (usually an alcohol modifier). The SFC×RPLC system of Francois, I. et al. employs a 2-position/10-port switching valve for the interface between the first dimension SFC unit and the second dimension RPLC unit. The system uses packed loops in the interface to prevent the analytes eluted from the SFC column from being forced into the waste line by the CO2 stream, and water is introduced into the loop to reduce interference of residual CO2 gas in the second dimension.
Cortes and co-workers (J. Microcol. Sep. 1992, 4:239-244; and U.S. Pat. No. 5,139,681) described a 2D LC×SFC system including a sample inlet capillary where volatile solvents from the first dimension LC is eliminated by passage of nitrogen gas leaving a deposit of the eluted analytes, which is then taken up by the CO2 mobile phase for the second dimension SFC. However, solvent elimination by passage of nitrogen gas is not practical for RPLC which uses aqueous mobile phase.
There remains a need for an efficient chromatography system and methods for separating and analyzing complex samples where it is advantageous to conduct a first dimension RPLC and a second dimension SFC separation.